(a) Field of the Invention
The invention relates to a culture medium which promote islet cells survival, which may be transplanted to reverse hyperglycemia.
(b) Description of Prior Art
Adequate numbers of isogeneic islets transplanted into a reliable implantation site can only reverse the metabolic abnormalities in diabetic recipients in the short term. In those that were normoglycemic post-transplant, hyperglycemia recurred within 3-12 months. The return of the diabetic state that occurs with time has been attributed either to the ectopic location of the islets, to a disruption of the enteroinsular axis, or to the transplantation of an inadequate islet cell mass.
Studies of the long term natural history of the islet transplant, that examine parameters other than graft function, are few in number. Only one report was found in which an attempt was specifically made to study graft morphology (Alejandro R. et al., J. Clin. Invest., 1986, 78:1339). In that study, purified islets were transplanted into the canine liver via the portal vein. During prolonged follow-up, delayed failures of graft function occurred. Unfortunately, the graft was only examined at the end of the study, and not over time as function declined. Delayed graft failures have also been confirmed by other investigators for dogs and primates. Most failures are presumed to be the result of rejection despite appropriate immunosuppression.
Because of these failures, there is currently much enthusiasm for the immunoisolation of islets, which could eliminate the need for immunosuppression. The reasons are compelling., Immunosuppression is harmful to the recipient, and may impair islet function and possibly cell survival. Unfortunately, micro-encapsulated islets injected into the peritoneal cavity of the dog fail within 6 months (Soon-Shiong P. et al., Transplantation, 1992, 54:769), and islets placed into a vascularized biohybrid pancreas also fail, but at about one year (Lanza RP et al., Diabetes, 1992, 41:1503). Histological evaluation indicates a substantial loss of islet mass in these devices. No reasons have been advanced for these changes.
Only whole pancreas transplantation is capable of permanently reversing the diabetic state and preventing the secondary complications of diabetes. This suggests that there is a fundamental biological difference between grafts of whole pancreas and those of purified islets.
It is perhaps significant that the major emphasis in islet transplantation has been the enhancement of the purity of the islet preparation to promote engraftment and reduce immunogenicity. After more than 20 years of concerted research and more than 200 attempts at human islet transplantation, the achievement of insulin independence remains elusive. Contamination of the islet preparation by non-endocrine cells does not impair function or engraftment, nor enhance immunogenicity. In a canine model, pancreatic tissue fragments autotransplanted into the spleen appeared to survive better than islets refluxed into the liver (Kretemom NM and Warnock GL, Transplantation, 1990, 49, 679). The survival advantage was attributed to the specific site, but pancreatic fragments may actually have an inherent survival advantage compared to purified islets. In people, insulin-independence has been obtained after intraportal injection of unpurified autologous islets. Fewer impure islets were more successful than many more purified ones. A similar experience was repeated with allotransplants, with unpurified islets from a single human pancreas successfully reversing hyperglycemia (Gores PF et al., Lancet, 1993, 341:19). From these and other reports, it is apparent that unpurified islets from one pancreas may survive as well as pure islets prepared from multiple donors.
To date, the only known roles of apoptosis, or programmed cell death, in the regulation of islet cell mass are in the involution of xcex2 cell mass after pregnancy and perhaps in remodeling during development as recently suggested by Finegood (Finegood OT et al., Diabetes, 1995, 44:249). These situations, however, are physiologic in nature and it remains to be determined under what unusual circumstances islets may also be induced to undergo apoptotic cell death. The recent report of a selective decrease in xcex2 cell mass following transplantation of human islets into diabetic nude mice is therefore of considerable interest. The induction of apoptosis in relation to islet isolation and transplantation has not been reported previously. We have original observations on human islets after isolation. Light microscopic examination of these islets just prior to culture demonstrates that at least 15% of the cells have morphological evidence of apoptosis (pyknotic nuclei). Tissue transglutaminase (TG) expression is a fundamental event in the induction of apoptosis. TG is a calcium-dependent enzyme whose activity is well established in many mammalian tissues, including pancreas. It is involved in the cross-linking of intracellular proteins that precedes the irreversible ultrastructural changes characterizing cells undergoing apoptosis. The appearance of apoptotic bodies parallels an increased expression of TG. Our pilot studies have shown TG activity to be elevated immediately following islet isolation, with continued increase up to at least 1 week. These findings correlated with data from a cell death ELISA that detects histone-associated DNA fragments, another characteristic of the apoptotic process (Paraskevas S. et al., Transplant Proc., 1997, 29:750). These fragments result from the activity of an endonuclease that cleaves DNA at internucleosomal sites. These data form the basis for investigating the role of apoptosis in islet survival after isolation.
It would be highly desirable to be provided with a means to prevent apoptosis of islet cells, thereby allowing transplantation with the successful reversal of hyperglycemia.
One aim of the present invention is to provide a culture medium which promotes the survival islet cell, which may be transplanted to reverse hyperglycemia.
In accordance with the present invention there is provided a medium for promoting the survival of islet cells, which comprises one or more growth factors in combination with FK506 in amounts having an anti-apoptotic effect on islet cells in a physiologically acceptable culture medium.
In accordance with a preferred embodiment of the present invention, a culture medium, herein referred to as duct-conditioned medium (DCM), is prepared from primary duct cultures that had been passaged twice to remove other cellular elements. DCM was analyzed for known growth factors. The presence of a large amount of IGF-II (34 ng/ml) and a much smaller quantity of NGF (4 ng/ml) were identified.
In accordance with another embodiment of the present invention there is provided a medium for promoting the survival of islet cells comprising, in a physiologically culture medium, IGF-I or IGF-II in combination with NGF in an amount having a synergistic anti-apoptotic effect on islet cells. More preferably, this medium further includes FK-506.
The preferred growth factors include, without limitation, NGF, IGF-II and insulin. Other growth factors include TGFxcex2, IGF-I and HGF.
The preferred NGF concentration is between about 10 to about 100 ng/ml of medium.
The preferred IGF-II concentration is between about 10 to about 100 ng/ml of medium.
Preferably, the culture medium of the present invention includes an immunosuppressant.
The preferred immunosuppressants are selected from the group consisting of FK506, a peptide having NGF potentiating effect, and therefore possibly a trophic effect on islet cells.
The preferred FK506 concentration is about 1 micromolar.
More preferably, the culture medium of the present invention further includes insulin. The preferred insulin concentration is from about 10 to about 100 ng/ml of medium.
More preferably, the culture medium of the present invention further includes a soluble matrix molecule called fibronectin and type I collagen.
The term xe2x80x9cphysiologically acceptable culture mediumxe2x80x9d is intended to mean any commercially available culture medium including, without limitation, CMRL 1066, RAM 1640 and DMEM/F12.
The culture medium of the present invention may also be used to isolate islet cells or to irrigate the site of transplant to promote in situ islet cells survival.